Several lines of indirect evidence support the conclusion that a TGA termination codon in the open reading frame of the gene for a formate dehydrogenase, fdhF, in Escherichia coli is used to direct the incorporation of selenocysteine in the gene product. However, direct proof that the TGA codon in the gene sequence corresponds to the location of the selenocysteine residue in the polypeptide was lacking. A fusion gene product containing a portion of the formate dehydrogenase surrounding the putative selenocysteine locus fused to beta-galactosidase was isolated by affinity chromatography and digested with endoproteinase Asp-N. A resulting aspartate N-terminal peptide that contained [75Se]selenocysteine was purified and subjected to Edman automated amino acid sequence analysis. 75Se released from the peptide in the 7th cycle corresponded to the position of selenocysteine predicted from the gene sequence. Exact sequence homology of the first 19 residues of the peptide with that predicted from the gene sequence confirmed that UGA directs selenocysteine incorporation in this E. coli protein. Based on the report of Arkowitz and Abeles [JACS, 1990] that the glycine reductase selenoprotein A is converted to its Se-carboxymethyl derivative during the reductive deamination of glycine, we have shown that the energy conserving step of the overall process involves the reductive cleavage of the selenoether derivative. Se[14C]carboxymethyl protein A was prepared directly by reaction with [14C]bromoacetate. When incubated with protein C (another enzyme component of the reductase system) in the presence of an added dithiol, the labeled carboxymethyl group was converted to an acetyl thiol ester derivative of protein C. Addition of phosphate converts the acetyl thiol ester to acetyl phosphate. The details of this novel reaction are investigated with pure selenoprotein A and pure protein C as reagents.